Manganese is an essential nutrient that can also be toxic, and exposure to environmental manganese can cause neurotoxicity. In spite of its public health importance, little is known regarding manganese homeostasis. The long-term goals of this program are to employ bakers' yeast as a model eukaryote to understand manganese metabolism and toxicity. The current proposal addresses three distinct but related aspects of manganese biology: (Aim 1) the sensing of manganese during manganese stress; (Aim 2) phosphate as a key determinant of manganese toxicity; and (Aim 3) the impact of mitochondrial iron on manganese binding to the anti-oxidant enzyme superoxide dismutase 2 (SOD2). In Aim 1, manganese stress is defined as extreme conditions of manganese starvation or manganese toxicity. Preliminary studies indicate that manganese sensing for these two stresses occurs in separate cell compartments, but in both cases, cells respond through post-translational control of a manganese transporter, Smf1p. Proposed studies will test the hypothesis that Smf1p itself is a sensor for manganese and will examine the mechanism by which Smf1p localization and expression are controlled by manganese stress. Regarding Aim 2, high intracellular phosphate was found to cause profound manganese toxicity in yeast, and this toxicity was reversed through chromatin remodeling factors. The possible epigenetic effects will be probed through yeast genetic screens and transcription profiling studies. Additionally, the profile of manganese-phosphate interactions inside the cell that accompany manganese toxicity will be obtained through 31P-NMR and ENDOR spectroscopy with help from expert collaborators. Finally, Aim 3 explores how a specialized pool of mitochondrial iron competes with manganese for binding to SOD2. Molecular genetics experiments will test the hypothesis that this SOD2-reactive iron is derived from the Fe-S biogenesis pathway, and by inhibiting manganese binding to SOD2, it contributes to mitochondrial damage in a disease of iron overload. The nature of SOD2-reactive iron will be evaluated through a colorimetric assay for mitochondrial ferrous iron and through XANES analyses (by our collaborator J. Penner-Hahn) of the metal coordination environment. Such investigations into SOD2-reactive iron will provide important clues as to how SOD2 normally selects manganese. Overall, this multi-disciplinary approach to understanding manganese ion biology should provide novel insight into the homeostasis of a toxic nutrient.